Fluorescence is a phenomenon of light emission by a substance that has previously absorbed light of a different wavelength. In most cases, the emitted light has a longer wavelength, and therefore lower energy, than the absorbed light. However, when the absorbed electromagnetic radiation is intense, it is possible for one atom or molecule to absorb two photons; this two-photon absorption can lead to emission of radiation having a shorter wavelength than the absorbed radiation. Fluorescent light can be easily separated from reflected excitation light, thus providing excellent selectivity in applications where fluorescent light may carry some useful information about the substances and structures which emitted it.
This property is particularly important in various medical imaging applications, where fluorescent light may be emitted by fluorescent dyes, also known as fluorophores, with affinity to certain biological materials such as blood, or dyes conjugated to biological markers with specific affinity to certain tissues, proteins or DNA segments, and can be a reliable proxy for imaging internal body structures, such as blood vessels, lymph nodes, etc., as well as finding signs of disease, such as necrosis or cancer.
Usually, fluorescent biological markers are introduced externally, specifically with a purpose of binding to and imaging specific organs and tissues. In some cases, they are naturally-occurring, which is known as biological auto-fluorescence.
Most fluorescent substances, whether biological or not, have specific absorption and emission spectra, with peaks at certain wavelength. Sometimes, more than one peak may be present in either absorption or emission spectrum, or both. In any case, any fluorescent imaging system must provide excitation light at one wavelength and detect the emission light at different wavelength. Since the optical efficiency of fluorescence is usually quite low, emission light is usually much weaker than excitation light. Hence, optical filters which accept emission light and block excitation light are also usually present in a fluorescent imaging system.
Of particular interest are the fluorescent dyes which both absorb and emit light in the Near Infrared (NIR) part of the spectrum, approximately from 700 to 1000 nm wavelength. Within this band, human tissues are particularly transparent, so the fluorescent dyes may be seen at most depths and images may be of particular clarity.
Fluorescent medical imaging systems are known in prior art, including those designed for detection of NIR fluorescent dyes.
Usually, fluorescent images are combined with conventional, reflected-light images and presented to a medical practitioner on a common monitor, so the distribution of the fluorescent die can be visible in its surroundings. Since the NIR fluorescent image is outside of the human visible light range, it is usually mapped to a human visible color and displayed on a monitor superimposed on top of the captured color image of the biological object of interest. The system can display either still or moving images. A medical practitioner can use such a system to detect, for example, cancer cells during surgery, detect perfusion during reconstructive surgery, and detect the location of lymph nodes. During open surgery, wherein the surgeon is directly viewing the field of surgery, utilization of a monitor is disadvantageous in the surgeon must glance away from the surgical site to view the image of the fluorescence. Upon returning to view the surgical area, the surgeon must estimate the position of the florescence based upon his memory of the display on the monitor. Alternative, the surgeon can perform the required work while directly viewing the monitor as opposed to directly viewing the surgical area. This approach is disadvantaged in that it is cumbersome to operate without directly viewing the surgical site. Further, when viewing the monitor the surgeon losses all three dimensional information that is normally obtained when directly viewing the surgical area.
While being valuable surgical and diagnostic tools, known fluorescent cameras suffer from a number of limitations, mostly stemming from very low signal levels produced by fluorescent dyes. Those limitations are insufficient sensitivity, low video frame rates or long integration time necessary for taking a still image, as well as a limited depth of focus, especially if a large objective lens is used to alleviate sensitivity problems.
There are many known fluorescent dyes and or molecules that are used in the medical field, also referred to florescent probes or fluorescent markers. (see, Thermo Scientific, Fluorescent Probes, available at: www.piercenet.com/browse.cfm?fldID=4DD9D52E-5056-8A76-4E6E-E217FAD0D86B, the disclosures of which are hereby incorporated by reference).
Furthermore, in the following article, near-infrared fluorescence nanoparticle-base probes for use in imaging of cancer are described and is hereby incorporated by reference: He, X., Wang, K. and Cheng, Z. (2010), “In vivo near-infrared fluorescence imaging of cancer with nanoparticle-based probes,” WIREs Nanomed Nanobiotechnol, 2: 349-366. doi: 10.1002/wnan.85.